Sulfitocobalamin and process of preparing the same



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SULFITOCOBALAMIN AND PROCESS OF PREPARING THE SAME Filed Sept. 5, 1952 2Sheets-Sheet l S E E 3 $s H. H. FRICKE Oct. 18, 1955 SULFITOCOBALAMINAND PROCESS OF PREPARING THE SAME Filed Sept. 5, 1952 2 Sheets-Sheet 25a 3 $623 a gm 8% QS 9R gm 3% 9% 9% 8m gm Q8 n W .M Z w( M /\1 Q9 w Qrfi:3 3 H Sm Q" S 9m ET 5 3% am? RS m6 gm m flmkm u\ mum M E m 5 9R 5::SEEmW zstmmcmmw United States Patent Ofiice 2,721,162 Patented Oct. 18,1955 SULFITOCOBALAMIN AND PROCESS OF PREPARING THE SAME Howard H.Fricke, Grayslake, 111., assignor to Abbott Laboratories, North Chicago,111., a corporation of Illinois Application September 5, 1952, SerialNo. 308,127

11 Claims. (Cl. 167-81) This invention relates to stable vitaminproducts and more particularly to a novel cobalamin derivative, namelysulfitocobalamin.

In the prior art an antipernicious anemia factor was isolated anddescribed by Rickes et al., Science, 107, 396, (1948); and Rickes etal., Science, 108, 134 (1948). Rickes et al. called the antiperniciousanemia factor vitamin B12, which name was subsequently adopted by U. S.Pharmacopoeia, 3rd sheet supplement to 13th volume of the U. S.Pharmacopoeia. Subsequently, Pierce et al., JACS, 71, 2952, (1949)isolated and described another compound having antipernicious anemiaactivity, and since the compound was very closely related to the vitamin1312 they called it vitamin B121). The two compounds show dift'erentabsorption spectrum maxima, which is a means used to identify the twocompounds. For instance, Brink et al., JACS, 71, 1954 (1949) showed theabsorption spectrum maxima for vitamin B12 to be at 2780 A. 3610 and5500 A. 10. Am whereas Pierce et al., supra, showed the absorptionspectrum maxima for vitamin B121; to be at 273,1, 351 1., and 525Vitamin B1 has been identified chemically as cyanocobalamin, and vitaminB12b has been identified as bydroxocobalamin.

Vitamin B12 and vitamin 1312?), as pure crystalline products in aqueoussolution are relatively stable, but as other ingredients are added tothe solution the vitamins may be destroyed. For instance, Gakenheimer etal., I. A. Pharm. Assoc., 28, 660 (December 1949), shows the vitamin B12is incompatible with ascorbic acid and is rapidly destroyed. It is nowknown that vitamin Bizb is even more rapidly destroyed by the additionof ascorbic acid to aqueous solutions thereof, and is almostinstantaneously destroyed by the addition of about mg. per cc. ofascorbic acid to aqueous solutions of vitamin B12! at about roomtemperature. Also, both vitamin B12 and vitamin Bizb are even morerapidly destroyed as the temperature of such solutions is increased.

The present application is a continuation in part of my copendingapplication, Serial No. 172,396, filed July 7, 1950, which was copendingwith my earlier application, Serial No. 118,197, filed September 27,1949 both now abandoned. In S. N. 172,396 there is described a stableaddition product obtained by adding sodium bisulfite to solutions ofvitamin B12 (cyanocobalamin) or vitamin B12b (hydroxocobalamin). InSerial No. 118,197 there is described a process for stabilizing vitaminB12 by adding a soluble bisulfite to solutions containing vitamin B12. Ihave now discovered that a new and separate entity, identified assulfitocobalamin, is formed when a soluble bisulfite such as sodiumbisulfite is added to a solution containing a cobalamin derivative suchas cyanocobalamin or hydroxocobalamin, in an amount in excess of thatrequired to form sulfitocobalamin with the cobalamin moiety present inthe solution. The resulting sulfitocobalamin shows a separate anddistinct absorption spectra as indicated in Figure 2. The absorptionmaxima for sulfitocobalamin under these conditions are 270-280;L,308310;r, 364-366 415422 and 516-540 1. A solution of sulfitocobalaminprepared from cyanocobalamiu is found to contain no cyanoradical.

Sulfitocobalamin is found to possess biological activity equivalent tothat of cyanocobalamin. In the presence of even a minute excess ofsulfite ions (for example, as little as 0.01%, sulfitocobalamin issubstantially more stable than cyanocobalamin in the presence ofascorbic acid in aqueous solution.

Referring to the drawings:

Figure 1 is a graph showing the absorption spectra of crystallinevitamin B12 (cyanocobalamin) and of vitamin 1312b (hydroxocobalamin);

Figure 2 is a graph showing the absorption spectrum of the product ofthis invention, sulfitocobalamin in aqueous solution in the presence ofsulfite ions.

The absorption spectrum curves of Fig. 1 show the slight difierences,and the overall similarity between cyanocobalamin and hydroxocobalamin.As noted above, the absorption spectrum of cyanocobalamin shows themaxima at 278g, 361 and 550,11. The absorption maxirna forhydroxocobalamin are 273 351,11, and 525 The spectrum curves of the twocompounds clearly show that the two compounds are different, thoughrelated.

A comparison of the spectrum curves of Fig. 1 and the spectrum curve ofFig. 2 clearly shows that sulfitocobalamin is a chemical entitydistinctly different from cyanocobalamin and hydroxocobalamin.

In more specific detail the following examples will illustrate theinvention:

Example I About 14.3 mg. of crystalline cyanocobalamin is dissolved in25 cc. of water. The solution shows absorption spectrum maxima at 278 1,361,11, and 550a (shown by the broken line in Fig. l). About 25 mg., orequivalent to 0.1% by weight, of sodium bisulfite is dissolved in thesolution, and the solution allowed to stand at room temperature forabout 24 hours. At the end of 24 hours a new chemical is formed havingabsorption spectrum maxima in solution at 270-280 364 and 540p. (shownby the curve of Fig. 2).

Sulfitocobalamin may be recovered from the mother liquor by extractionwith a suitable solvent such as methanol. The residue after evaporationof the methanol may be dissolved in water, and the solution thus formedmay be passed over a column of ion exchange resins (Amberlite IR- andAmberlite IR4B). The ion exchange resins remove the sulfito radical andexchange it for the hydroxo-radical, thereby producing hydroxocobalamin.The solution recovered from the column is concentrated under vacuum toabout 1 cc., and about 6 cc. of acetone is added to the solution whichcauses hydroxocobalamin to crystallize. An aqueous solution of thecrystallized hydroxocobalamin gives the characteristic absorptionspectrum curve of hydroxocobalamin shown in the solid curve of Fig. 1.

Example II Among 0.485 mg. of crystalline cyanocobalamin dissolved inabout 20 cc. of water, gives absorption spectrum maxima at 278,11, 361a,and 549a.

About 0.500 mg. of crystalline hydroxocobalamin dissolved in 20 cc. ofwater, gives absorption spectrum maxima at 273a, 352,11, 411,14, and527a.

About 20 mg. or about 0.1% by weight, of sodium bisulfite is added toeach of the above solutions, and after standing for about 72 hours atroom temperature, the absorption spectrum maxima of the two solutions isas follows:

The solution which originally contained cyanocobal- 3 amin now showsabsorption spectrum maxima at 275 364-365 t, 4l6418,u., and 516g.

The solution which originally contained hydroxocobalamin now showsabsorption spectrum ,maxima at 275p, 365p, 418-422 and 516 Within iexperimental error the absorption spectrum maxima of the two solutionsshow that cyanocobalamin and hydroxobalamin each form sulfitocobalaminwhen treated'with sodium bisulfite in excess.

In the examples above, the soluble bisulfite used is sodium bisulfite,but any water-soluble bisulfite salt may be used. In place of the sodiumbisulfite, other alkali metal and alkali earth metal bisulfites maybeused, such as potassium, lithium, calcium, magnesium, etc. Othersuitable salts are the nitrogen salts including the ammonium salt aswell as the substituted ammonium salts including the primary, secondary,and tertiary alkyl and substituted alkylamine bisulfites and thecorresponding ammonium bisulfites. The alkyl groups include methyl,ethyl, propyl, butyl, etc., amine salts, as well as the correspondinghydroxyalkyl or alkanol amine salts. The phenyl alkylamines, e. g.,benzylamine, as well as other aromatic amines, e. g., aniline, may alsobe employed. While, as indicated above, any water-soluble bisulfite saltmay be used, it is preferable not to use salts of relatively hightoxicity, such as barium bisulfite, etc. In the final solution, thesulfitocobalamin should be prepared with a non-toxic soluble bisulfite,as such solutions are generally injectable therapeutic products.

Others may readily adapt the invention for use under various conditionsof service, by employing one or more of the novel features disclosed orequivalents thereof. As at present advised with'respect to the apparentscope of my invention, I desire to claim the following subject matter. I

I claim:

1. The compound sulfitocobalamin characterized b absorption spectrummaxima at 270-280 1, 364-366;, 415-422 and 5 16-540 1.

2. The process of preparing sulfitocobalamin which comprises, intimatelycontacting a cobalamin derivative selected from the class consisting ofcyanocobalamin and hydroxocobalamin with a water-soluble bisulfite saltin excess of the amount required to react with the cobalamin moietypresent in the solution.

3. A process according to claim 2 in which the bisulfite salt is presentto the extent of about 0.01% to about 5.0%.

4. The process of preparing sulfitocobalamin which comprises, intimatelycontacting an aqueous solution of cyanocobalamin with a water-solublebisulfite salt selected from the group consisting of alkali metal,alkaline earth, and amine salts in excess of'that amount required toreact with the cobalamin moiety present in the solution. a

5. The process of preparing sulfitocobalamin which comprises, intimatelycontacting an aqueous solution of hydroxocobalamine with a water-solublebisulfite salt selected from the group consisting of alkali metal,alkaline earth, and amine salts in excess of that amount required toreact with the cobalamin moiety present in the solution.

6. The process of preparing sulfitocobalamin which comprises, intimatelycontacting an aqueous solution of a cobalamin derivative selected fromthe class consisting of cyanocobalamin and hydroxocobalamin with sodiumbisulfite in excess of that amount required to react with the cobalaminmoiety present in the solution.

7. The process of preparing sulfitocobalamin which comprises, intimatelycontacting an aqueous solution of a cobalamin derivative selected fromthe class consisting of cyanocobalamin and hydroxocobalamin withpotassium bisulfite in excess of that amount required to react with thecobalamin moiety present in the solution.

8. The process of preparing sulfitocobalamin which comprises, intimatelycontacting an aqueous solution of cyanocobalamin with sodium bisulfitein excess of that amount required to react with the cobalamin moietypres-' ent in the solution.

9. The process of preparing sulfitocobalamin which comprises, intimatelycontacting an aqueous solution of hydroxocobalamin with sodium bisulfitein excess of that amount required to react with the cobalamin moietypresent in the solution.

10. The process of preparing sulfitocobalamin which comprises,intimately contacting an aqueous solution of a cobalamin derivativeselected from the class consisting of cyanocobalamin andhydroxocobalamin with ammonium bisulfite in excess of that amountrequired to react with the cobalamin moiety present in the solution.

11. A stable, aqueous solution containing sulfitocobalamin as an activeingredient, and an excess of sulfite OTHER REFERENCES Merck Index, 5thedition (1940), pg. 506.

Rusby, Bliss and BallardProperties and Uses of Drugs (1930), pg. 506.

U. S. Dispensatory, 24th Ed., (1947), pg. 1588;

GirdwoodLancet, Aug. 20, 1949, pg. 346.

StokstadFederation Proceedings (American Society of BiologicalChemists), March 1949, pg.257.

Amber-hi-Lites, published by Rohm & Haas, Number 3, August 1949, firstpage.

PierceJournal of the American Chemical Society,

vol. 71, August 1949, pg. 2952.

Brockman]ournal of the American Chemical Society, vol. 72, February1950, pg. 1042. 1

1. THE COMPOUND SULFITOCOBALAMIN CHARACTERIZED BY ABSORPTION SPECTRUMMAXIMA AT 270-280U, 364-366U, 415-422U, AND 516-540U.